Neoplasia is a disease characterized by an abnormal proliferation of cell growth known as a neoplasm. Neoplasms may manifest in the form of a leukemia or a tumor, and may be benign or malignant. Malignant neoplasms, in particular, can result in a serious disease state, which may threaten life. Significant research efforts and resources have been directed toward the elucidation of antineoplastic measures, including chemotherapeutic agents, which are effective in treating patients suffering from neoplasia. Effective antineoplastic agents include those which inhibit or control the rapid proliferation of cells associated with neoplasms, those which effect regression or remission of neoplasms, and those which generally prolong the survival of patients suffering from neoplasia. Successful treatment of malignant neoplasia, or cancer, requires elimination of all malignant cells, whether they are found at the primary site, have extended to local/regional areas, or have metastasized to other regions of the body. The major therapies for treating neoplasia are surgery and radiotherapy (for local and local/regional neoplasms) and chemotherapy (for systemic sites).
Despite the various methods for detecting, diagnosing, and treating cancers, the disease remains prevalent in all segments of society, and is often fatal. Clearly, alternative strategies for detection (including the development of markers that can identify neoplasias at an early stage) and treatment are needed to improve survival in cancer patients. In particular, a better understanding of tumor suppressors, and tumor-suppression pathways, would provide a basis from which novel detection, diagnostic, and treatment regimens may be developed.
The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress (Levine, A. J., Cell 88:323-31, 1997; Oren, M., J. Biol. Chem. 274: 36031-034, 1999). p53 can be thought of as the central node of a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage responses (e.g., ATM/ATR activation), abnormal oncogenic events (e.g., Myc or Ras activation) and everyday cellular processes (e.g., growth factor stimulation). While p53 mutations have been well documented in more than half of all human tumors (Hollstein et al., Mutat Res. 431:199-209, 1999), defects in other components of the p53 pathway, such as the ARF tumor suppressor, are observed in tumor cells that retain wildtype p53 (Sherr, C. J., Nat Rev Mol Cell Biol 2:731-737, 2001; Sharpless, N. E., DePinho, R. A., J Clin Invest 113:160-8, 2004). Activation of the p53 pathway appears to be a common, if not universal, feature of human cancer.
The mechanisms of p53 activation are not fully understood, but are generally thought to entail post-translational modifications, such as ubiquitination, phosphorylation and acetylation. Ubiquitination of p53 was first discovered in papilloma-infected cells, where p53 degradation is mediated by the viral E6 protein and a HECT-domain containing ubiquitin ligase called E6-AP (Munger, K., Howley, P. M., Virus Res 89:213-228, 2002). In normal cells, Mdm2 acts as a specific E3 ubiquitin ligase for p53, which, if malignantly activated, has the potential to counteract the tumor suppressor activity of p53. The critical role of Mdm2 in regulating p53 is illustrated by studies carried out in mice where inactivation of p53 was shown to completely rescue the embryonic lethality caused by loss of Mdm2 function (Montes de Oca Luna, R., Wagner, D. S., Lozano, G., Nature 378:203-206, 1995).
Although earlier studies suggested that Mdm2 is the primary factor in controlling p53 stabilities, the degradation of p53 is more complex than originally anticipated. The present inventor found that Mdm2 differentially catalyzes either monoubiquitination and polyubiquitination of p53 in a dosage-dependent manner (Li, M., Brooks, C. L., Wu-Baer, F., Chen. D., Baer, R., Gu, W., Science 302:1972-1975, 2003). Low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote polyubiquitination and nuclear degradation of p53. These mechanisms are exploited in different physiological settings. On one hand, Mdm2-mediated polyubiquitination and nuclear degradation may play a dominant role in suppressing p53 function when Mdm2 is malignantly overexpressed or during the late stages of a DNA damage response. On the other hand, Mdm2-mediated monoubiquitination and subsequent cytoplasmic translocation of p53 may represent an important means of p53 regulation in unstressed cells, where Mdm2 is maintained at low levels (Li et al., 2003, supra). Moreover, additional cellular factors may be necessary to facilitate p53 degradation, particularly when endogenous Mdm2 activities are not sufficient to catalyze p53 polyubiquitination directly. It was recently reported that ubiquitin ligases COP1 and Pirh2 are directly involved in p53 degradation (Dornan, D., Wertz, L, Shimizu, H., Arnott, D., Frantz, G. D., Dowd, P., O'Rourke, K., Koeppen, H., Dixit, V. M., Nature 429:86-92, 2004). Therefore, while Mdm2 is a key regulator of p53 function, p53 degradation acts through both Mdm2-dependent and Mdm2-independent pathways in vivo.
ARF (known as p14ARF in humans and p19ARF in mouse) was identified as an alternative transcript of the Ink4a, ARF tumor suppressor locus, a gene that encodes the p16Ink4a inhibitor of cyclin-dependent kinases. By virtue of its unique first exon, the ARF transcript encodes a protein that is unrelated to p16Ink4a. Nevertheless, ARF, like p16Ink4a, exhibits tumor suppression functions, as demonstrated by the tumor susceptibility phenotype of p14ARF-deficient mice. ARF suppresses abherrant cell growth in response to oncogene activation, at least in part, by inducing the p53 pathway (Sherr, et al., 2001, supra). The ARF induction of p53 appears to be mediated through Mdm2, since overexpressed ARF interacts directly with Mdm2 and inhibits its ability to promote p53 degradation (Zhang, Y., Xiong, Y., Yarbrough, W. G., Cell 92:125-34, 1998). The mechanisms by which ARF modulates the Mdm2/p53 pathway appears to be complex, both stabilizing p53 by binding and sequestering Mdm2 and activating p53 function by directly inhibiting the ubiquitin ligase activity of Mdm2.
Interestingly, ARF also has tumor suppressor functions that do not depend on p53 or Mdm2. For example, although ARF can induce cell growth arrest in tumor cells that lack a functional p53 gene (Normand, G., Hemmati, P. G., Verdoodt, B. et al., J. Biol Chem 280:7118-30, 2005) or a gene encoding the p21 cyclin-dependent kinase inhibitor, a key transcriptional target of p53, ARF can also suppress the proliferation of MEFs lacking both Mdm2 and p53. Consistent with these findings, the tumor susceptibility of triple knockout mice that lack ARF, p53 and Mdm2 is significantly greater than that associated with mice lacking any one of these genes alone. It was recently shown that ARF suppresses the growth, progression, and metastasis of mouse skin carcinomas through both p53-dependent and p-53 independent pathways (Kelly-Sprat, K. S., Gurley, K. E., Yasui, Y., Kemp, C. J., PLoS Biol. 2:E242, 2004). Distinct downstream factors may exist that mediate the p53-independent functions of ARF. The identity of these factors and the mechanisms by which they mediate p53-independent tumor suppression by ARF are unknown. Accordingly, while regulation of the p53 pathway is of intense interest and presents a potential means of diagnosing and treating cancers, a greater understanding of this pathway and the factors and mechanisms that mediate the p53 independent functions of ARF would provide a valuable basis upon which new diagnostic and therapeutic methods may be developed.